340 DANIEL I. ARNON 



but also, the final products of this process, starch and molecular oxygen, 

 are formed in or at the surface of illuminated chloroplasts. 



Chloroplasts were once widely believed to be the site of complete 

 photosynthesis, that is, of oxygen evolution coupled with carbon dioxide 

 assimilation [6, 7]. But this view was not supported by critical experi- 

 mental evidence and was later abandoned when Hill found in 1937 that 

 isolated chloroplasts produce oxygen in light but cannot assimilate CO., 

 [8-12]. Investigators who followed Hill corroborated his statement that 

 " if we break the green cell, it is possible to separate the fluid containing 

 the chloroplast and chloroplast fragments from the tissue residue. This 

 green juice can no longer assimilate CO^ but in the case of many plants 

 the insoluble green material, for a time at least, is still capable of giving 

 oxygen in light" [8]. 



In 1954 we found that previous difficulties in obtaining CO2 fixation 

 by isolated chloroplasts were indeed methodological. By using gentler 

 techniques of isolating chloroplasts from leaves, we prepared spinach 

 chloroplasts that were capable not only of giving the expected Hill reaction, 

 i.e. oxygen evolution, but also of converting COo to starch and sugar at 

 physiological temperatures and with no energy supply except visible 

 light [13-15]. 



Under the new experimental conditions, COo assimilation by isolated 

 chloroplasts was strictly light-dependent and proceeded at an almost 

 constant rate for at least an hour. There was approximate correspondence 

 between the oxygen evolved and the COo fixed, as would be expected from 

 the well-known photosynthetic quotient in green plants, O2/CO2 = i. The 

 products of CO2 assimilation were found to be the same as in photosyn- 

 thesis by whole cells. The insoluble product of CO2 fixation by chloro- 

 plasts was identified as starch. Among the soluble products the following 

 were found : phosphate esters of fructose, glucose, ribulose, sedoheptulose, 

 dihydroxyacetone, and glyceric acid; glycolic, malic, aspartic acids, 

 alanine, glycine and free dihydroxyacetone and glucose [14, 15]. Using 

 similar techniques, investigators in several different laboratories have 

 confirmed the ability of illuminated chloroplasts to form starch and sugars 

 from CO2 and water [cf. 16-21]. 



Most of the early work on extracellular photosynthesis was done with 

 spinach chloroplasts. But more recently, the same products of CO2 

 assimilation in light were also obtamed with isolated chloroplasts from 

 several diflerent species : sugar beet, sunflower, Phytolacca americana and 

 Tetragonia expansa [22, 23]. 



There was thus finally a firm experimental basis for concluding that 

 chloroplasts are indeed the sites of complete photosynthesis in green 

 plants. In the light of the new evidence, chloroplasts emerged as remark- 

 ably complete and autonomous cellular structures that have become 



